The present invention generally relates to reagents that are essential for an enzymatic reaction and that enhance reaction-product analysis. In specific, preferred embodiments, the invention provides compositions of essential components which facilitate subsequent chromatographic or electrophoretic analysis.
Enzymes are frequently used in laboratories to catalyze a variety of transformations. Typical enzymes which have been utilized include proteases, peroxidases, oxidases, kinases, amylases, and several nucleic acid modifying enzymes such as DNA polymerases, RNA polymerases, ligases, kinases, restriction endonucleases, phosphodiesterases, DNases, exonucleases, RNases, and phosphatases. The nucleic acid-modifying enzymes have been frequently used in molecular biology laboratories as part of procedures such as polymerase chain reaction (“PCR”), sequencing, southern hybridization analysis, restriction endonuclease analysis, RNase protection, and the production of labeled probes.
The steps involved in performing an enzyme catalyzed transformation can generally be categorized as reaction mixture formulation, enzymatic reaction, reaction product characterization, and reaction product use. The steps of mixture formulation, product characterization, and product use are labor intensive. The formulation of enzyme reaction mixtures entails combining reaction components which are essential for the enzymatic reaction into a reaction mixture. The reaction mixture is then incubated under conditions favorable for the enzymatic reaction to take place, and for a time sufficient to allow the enzymatic reaction to proceed substantially to completion. The reaction mixture is typically analyzed to evaluate the characteristics of the products formed. This analysis often entails a chromatographic or electrophoretic procedure to separate and evaluate the reaction products, and to determine whether the enzymatic reaction has proceeded to completion. Downstream applications entail a wide variety of varied uses for the products of enzymatic reactions, such as utilization in manufacturing, and further processing of the product with enzymes or chemical processes. In the case of molecular biological enzymatic reactions, examples of downstream applications are transformation of prokaryotic or eukaryotic cells, detection of complementary sequences by southern or northern hybridization, sequencing, phosphorylation, dephosphorylation, ligation, restriction digestion, endonucleolytic digestion, exonucleolytic digestion, and purification.
Procedures such as liquid chromatography and polyacrylamide gel electrophoresis (“PAGE”) have been frequently used to analyze the results of the enzymatic reactions by separating the reaction products by, for example, molecular weight. The results of the modification of nucleic acids by enzymes such as DNA polymerase have typically been analyzed by subjecting the reaction products to electrophoresis through polyacrylamide or agarose gels.
To analyze enzymatic reaction products using chromatography or electrophoresis, the sample to be analyzed has often been combined with components which assist the operator in performing the separation. One such component is a “tracer”, which is a detectable moiety such as a dye which is generally added to the sample immediately before loading the sample onto the chromatography column or electrophoresis gel. The tracer migrates in the medium in the same direction as the sample to indicate the progress of the separation.
Another reagent, termed “high density agent” herein, has also been commonly utilized in electrophoretic analysis of the enzymatic reaction products. High density agents are generally water soluble, dense liquids, such as a solution of sucrose or glycerol, which have been mixed with the sample, usually after the enzymatic reaction is complete, to increase the sample density. The increased density of the sample resulting from mixing the sample with the high density agent aids, for example, in loading the sample into a well of an electrophoresis gel by allowing the sample, when pipetted into the top of the well, to “fall” through the less-dense electrophoresis buffer solution to the bottom of the well.
High density agent and tracer have been combined with reaction product-containing samples to be electrophoretically separated. The combination of high density agent and tracer is generally termed “loading buffer”.
While tracers and loading buffers have usually been mixed with the sample after the enzymatic reaction is complete, as diagramed in FIG. 1, their use before the commencement of nucleic acid-modifying enzymatic reactions is also known, as diagramed in FIG. 2. Hoppe et al., BioTechniques 12: 679–680 (1992) describe combining a solution of sucrose (up to 30%) and certain dyes (cresol red, tartrazine, or yellow food coloring #5) with an enzyme reaction mixture containing all other components for PCR. After the PCR procedure, the samples were reportedly loaded directly onto an agarose gel for electrophoretic analysis. The authors noted that several dyes and heavy components were inhibitory to the Taq polymerase enzyme used, but that sucrose, cresol red, tartrazine, and yellow food coloring #5 were compatible with Taq.
Certain commercially available products provide tracer or loading buffer for use in enzyme reaction mixes for PCR. A thermostable polymerase preparation, Red Hot DNA Polymerase, is available from Advanced Biotechnologies and reportedly contains a red dye for use to indicate enzyme addition in the enzyme reaction mixture. There are also two products available which comprise a red tracer and a high density agent, for addition to a PCR reaction mixture before amplification. One, called RediLoad, is available from Research Genetics, Inc., and the other, called Rapid-Load™, is available from OriGene Technologies. These reagents must be added to the reaction mixture in a separate pipetting step.